Polypeptides derived from rna polymerases and use thereof

ABSTRACT

Mutant RNA polymerases of phagic origin in which the peptide chain is modified by substitution, deletion or addition of at least one amino acid, the modification having the effect of reducing the sensitivity of the RNA polymerases to the initial transcription sequence of the DNA sequence coding for the RNA, for a method for production of the RNA, or proteins coded by the RNA, from given nucleotide sequences, comprising a sequence of DNA coding for the RNA, the transcription of which is placed under the control of a promoter recognised by wild-type RNA polymerases and the mutant RNA polymerases as above. The method has a higher yield of RNA than the yield obtained when using the wild-type RNA polymerases in the presence of the same non-consensual ITS as that found in the sequence of DNA coding for the RNA.

[0001] A subject of the present invention is polypeptides derived fromRNA polymerases, also designated mutated RNA polymerases, as well astheir uses, in particular in the field of in vivo or in vitropreparation of RNA of interest.

[0002] When obtaining large quantities of DNA molecules of perfectlydefined sequence, high purity and small size (10 to 100 nt) chemicalsynthesis is at present the most economical method. There are now in themarket a very large number of enterprises which carry out thesesyntheses on request. These same companies also offer the chemicalsynthesis of RNA but the technology is such that the cost, which is atleast ten times higher than for DNA, is at present prohibitive.

[0003] Enzyme synthesis is therefore favoured, using an RNA polymerasecoupled to a DNA matrix, chemically synthesized or of plasmid origin,which comprises the sequence to be transcribed downstream of thepromoter sequence of the polymerase used. The necessary elements(polymerases, rNTP, cloning vectors) are available on the market in theform of optimized kits.

[0004] Bacteriophage T7 RNA polymerase is very widely used for carryingout transcription reactions in vitro with the aim of synthesizing largequantities (up to a milligram) of RNA from matrices of recombinant DNA.These RNAs synthesized in vitro are necessary for a number of uses inBiology, the Biotechnologies and Pharmacy; the following can bementioned inter alia:

[0005] translation in vitro.

[0006] the study of RNA maturation.

[0007] the effect of anti-sense RNA on gene expression.

[0008] RNA-protein interactions.

[0009] the synthesis of homologues of small cell RNAs (tRNA, rRNA).

[0010] the production of ribozymes.

[0011] More generally these RNAs are also used as molecular probes andstructural study substrates.

[0012] A few T7 polymerase mutants have already been described. Theseare mutated RNA polymerases derived from the wild-type T7 RNApolymerase, and comprising one of the following mutations:

[0013] replacement of the lysine (K) in position 222 by glutamic acid;this RNA polymerase thus mutated has the property of recognizing apromoter altered by mutation, and is described in U.S. Pat. No.5,385,834,

[0014] replacement of the tyrosine (Y) in position 639 by aphenylalanine (F); this RNA polymerase thus mutated has the property ofincorporating deoxyribonucleotides instead of ribonucleotides, thusallowing the synthesis of DNA instead of RNA, and is described in U.S.Pat. No. 6,107,037,

[0015] replacement of the isoleucine (I) in position 810 by a serine(S); this RNA polymerase thus mutated has the property of being slowerthan the wild-type RNA polymerase, and is described in Bonner et al.,The Journal of Biochemical Chemistry, 269, pp. 25120-25128 (1994).

[0016] The problem to be resolved by the present invention, is linkedwith the fact that the activity on a given DNA matrix of the T7 RNApolymerase (and moreover of all the other known RNA polymerases) isstrongly dependent on the nature of the first 6-12 nucleotidestranscribed (Initial Transcribed Sequence, or ITS) of the nucleotidesequence coding for a given RNA (Milligan et al., 1987). If the ITSdiffers too much from the consensus sequence 5′ GGGAGA . . . then thepolymerase frequently aborts and a large quantity of the ribonucleosidetriphosphates is consumed in order to synthesize small abortive RNAs tothe detriment of the desired large RNAs.

[0017] Therefore, the experimenter is generally compelled, in order toobtain an effective transcription, to modify the sequence that is to betranscribed, in order to add to it a favourable ITS (also referred to asconsensus or consensual ITS). In a number of cases, this constraint isvery annoying, even unacceptable.

[0018] A potential method for obtaining any non-consensual 5′ end RNAuses an after treatment of an RNA comprising the desired sequencedownstream of a consensus ITS; the latter (target) RNA can therefore beproduced in abundance. Moreover it is necessary to chemically synthesizea chimeric DNA-RNA oligonucleotide the DNA sequence of which iscomplementary to the 3′ end of the RNA to be eliminated, then the hybridthat can form the oligo with the target transcript is digested by RNaseH. This complex and onerous method is justified if the desired RNA is ofa very large size and if its 5′ end must be rigorously defined. (Li etal. 1999, Lapham et al. 1997).

[0019] The addition, to the reaction medium, of synthetic polyamines canincrease the production of RNA by, it appears, reducing the number ofabortive cycles; however determination of the optimum experimentalconditions (choice of polyamine, concentration of use) requirescase-by-case research. Moreover it should be noted that the effect ofthese polyamines is weak, even non-existent, when the matrix is ofplasmid origin (double strand) (Frugier et al. 1994).

[0020] These two methods use reagents which are not in common use(specific polyamines or oligonucleotides) and are unsuitable for routineuse. It is evident that the possibility of having, at the start, apolymerase which by nature aborts less would be a much simpler way ofresolving the difficulty.

[0021] The invention results from the demonstration by the Inventors ofthe fact that modifying the peptide sequence of the RNA polymerasescould reduce the sensitivity of the RNA polymerases thus modified to thenature of the ITS of the nucleotide sequences to be transcribed.

[0022] The invention aims to provide polypeptides derived from wild-typeRNA polymerases usually used in the production of RNA of interest, whichare distinctly less sensitive to the nature of the non-consensual ITSthan said wild-type RNA polymerases.

[0023] The invention also aims to provide new processes for producingRNA of interest with yields greater than those of the processes usingwild-type RNA polymerases when the latter are sensitive to the ITS ofthe nucleotide sequence coding for said RNA of interest.

[0024] A subject of the invention is the use of mutated RNA polymerasesof phage origin, namely of RNA polymerases originating from phages, thepeptide chain of which is modified, with respect to the wild-type RNApolymerases from which they derive, by substitution, or deletion, oraddition of at least one amino acid, this modification having the effectof reducing the sensitivity of said RNA polymerases to the ITS containedin the DNA sequence coding for an RNA of interest, for theimplementation of a process for producing said RNA of interest, or ofproteins coded by this RNA of interest, starting with determinednucleotide sequences comprising a DNA sequence coding for said RNA ofinterest and the transcription of which is placed under the control of apromoter recognized by the abovementioned wild-type RNA polymerases andmutated RNA polymerases, said process having a production yield of saidRNA greater than the yield obtained in the case of use of the wild-typeRNA polymerases (in the presence of the same non-consensual ITS as thatcontained in the DNA sequence coding for said RNA of interest).

[0025] The activity of said RNA polymerases thus mutated within thetranscription of the DNA sequence coding for said RNA of interest isdistinctly less affected by the nature of the nucleotides constitutingthe non-consensual ITS of this DNA sequence, which is not that of thewild-type RNA polymerases from which they derive, which allows thesemutated RNA polymerases to be up to approximately 40 times more activethan the wild-type polymerases, and therefore, in the abovementionedprocess of the present invention, to be characterized by a productionyield of said RNA greater than the yield obtained in the case of use ofthe wild-type RNA polymerases in the presence of this samenon-consensual ITS.

[0026] According to another particularly advantageous aspect of theinvention, the mutated RNA polymerases of the invention make it possibleto obtain RNA of interest with a practically identical yield, whateverthe ITS present in the sequence coding for said RNA of interest.

[0027] Advantageously, the use of the abovementioned mutated RNApolymerases allows the implementation of a process for producing RNA ofinterest, the yield of which is up to approximately ten times greaterthan the yield obtained in the case of use of the wild-type RNApolymerases in the presence of a non-consensual ITS.

[0028] The RNA of interest capable of being produced in greater quantitywithin the implementation of a process according to the invention usingthe abovementioned mutated RNA polymerases, are natural or chimericRNAs, said RNAs if appropriate comprising one or more non-canonicalnucleoside monophosphates (namely for example a deoxyribose instead of aribose, said sugar itself being able optionally to carry an analogue ofone of the natural nucleic bases if this analogue is recognized as suchby the polymerase). In the latter case, a subject of the presentinvention is also the use of the above-mentioned process using saidabove-mentioned RNA polymerases, in the implementation of a method fordetermining the sequence of a nucleic acid molecule.

[0029] The mutated RNA polymerases used are advantageously thosederiving from wild-type phage monomeric polymerases, in particular thoseoriginating from monomeric RNA polymerases of bacteriophages such as T7,T3, K11, SP6, respectively described in particular in the article by W.T. McAllister and C. A. Raskin, Molecular Microbiology (1993), 10(1),1-6.

[0030] Preferably the abovementioned mutated RNA polymerases are thosederiving from the wild-type RNA polymerases at least one of the aminoacids of which, situated between positions 1 and approximately 410, inparticular approximately between positions 90 and 320, more particularlybetween positions 115 and 300, is modified by substitution or deletion.

[0031] Advantageously, the mutated RNA polymerases used are thosecomprising a leucine in position 266, substituted for the prolinesituated in position 266 in the wild-type T7 RNA polymerase, or theproline situated in homologous position in the wild-type RNA polymerasesof bacteriophages, such as the proline situated in positions 267 in T3,289 in K11, and 239 in SP6.

[0032] A more particular subject of the invention is the abovementioneduse of any mutated RNA polymerase as defined above, the proline ofwhich, defined above, and/or at least one of the amino acids situated inthe vicinity of the abovementioned proline, namely an amino acidsituated at a distance less than or equal to approximately 10 angströmsfrom the proline in question, when said RNA polymerase is considered inits three-dimensional structure (as described in Cheetham, G. M. &Steitz, T. A. (1999), Science 286, 2305-2309; Cheetham, G. M. et al.,Nature 399, 80-83; Sousa, R. et al., Nature 364, 593-599), is modifiedby substitution or deletion.

[0033] The invention also relates to the abovementioned use of theparticularly preferred mutated RNA polymerases chosen from thefollowing:

[0034] those derived from the wild-type T7 RNA polymerase, andcomprising at least one of the following mutations:

[0035] replacement of the isoleucine (I) in position 117 by a valine(V),

[0036] replacement of the isoleucine (I) in position 119 by a valine(V),

[0037] replacement of the valine (V) in position 134 by an alanine (A),

[0038] replacement of the aspartic acid (D) in position 147 byasparagine (N),

[0039] replacement of the histidine (H) in position 230 by an arginine(R),

[0040] replacement of the proline (P) in position 266 by a leucine (L),

[0041] replacement of the arginine (R) in position 291 by a cysteine(C),

[0042] those derived from the wild-type T3 RNA polymerase, andcomprising at least one of the following mutations:

[0043] replacement of the aspartic acid (D) in position 148 byasparagine (N),

[0044] replacement of the proline (P) in position 267 by a leucine (L),

[0045] replacement of the arginine (R) in position 292 by a cysteine(C),

[0046] those derived from the wild-type K11 RNA polymerase, andcomprising at least one of the following mutations:

[0047] replacement of the aspartic acid (D) in position 167 byasparagine (N),

[0048] replacement of the proline (P) in position 289 by a leucine (L),

[0049] replacement of the arginine (R) in position 314 by a cysteine(C),

[0050] those derived from the wild-type SP6 RNA polymerase, andcomprising at least one of the following mutations:

[0051] replacement of the aspartic acid (D) in position 117 byasparagine (N),

[0052] replacement of the proline (P) in position 239 by a leucine (L).

[0053] A more particular subject of the invention is the abovementioneduse of the following mutated RNA polymerases:

[0054] the mutated T7 RNA polymerase represented by SEQ ID NO: 2,comprising a leucine in position 266 substituted for the proline,

[0055] the mutated T7 RNA polymerase represented by SEQ ID NO: 4,comprising a valine in position 117 substituted for the isoleucine, andan alanine in position 134 substituted for the valine,

[0056] the mutated T7 RNA polymerase represented by SEQ ID NO: 6,comprising a valine in position 119 substituted for the isoleucine, andan asparagine in position 147 substituted for the aspartic acid,

[0057] the mutated T7 RNA polymerase represented by SEQ ID NO: 8,comprising an arginine in position 230 substituted for the histidine,and a cysteine in position 291 substituted for the arginine,

[0058] the mutated T7 RNA polymerase represented by SEQ ID NO: 10,comprising a leucine in position 266 substituted for the proline, and aphenylalanine in position 639 substituted for the tyrosine,

[0059] the mutated T7 RNA polymerase represented by SEQ ID NO: 12,comprising an asparagine in position 810 substituted for the isoleucine,

[0060] the mutated T7 RNA polymerase represented by SEQ ID NO: 14,comprising a leucine in position 266 substituted for the proline, and anasparagine in position 810 substituted for the isoleucine,

[0061] the mutated T7 RNA polymerase represented by SEQ ID NO: 16,comprising a valine in position 119 substituted for the isoleucine, anasparagine in position 147 substituted for the aspartic acid, and anasparagine in position 810 substituted for the isoleucine.

[0062] A subject of the invention is also the use described above ofabovementioned mutated RNA polymerases comprising as well as themodification(s) of amino acid(s) defined above, the modification, bysubstitution, or deletion, or addition, of at least one other amino acidinvolved in a mechanism other than that of sensitivity to the ITS withinthe scope of the transcription of DNA sequences, such as a modificationof an amino acid making it possible for the RNA polymerase thus modifiedto incorporate non-canonical nucleoside triphosphates (for example dNTP)in an RNA chain, and therefore making it possible to synthesize DNA,and/or a modification of an amino acid making it possible to obtain aslower RNA polymerase, and therefore, in association with a mutation ofthe invention, making it possible to improve the synthesis yields ofproteins of interest.

[0063] A more particular subject of the invention is therefore theabovementioned use of mutated RNA polymerases as defined above,comprising as well as the mutation(s) defined above, a mutationcorresponding to the substitution of the tyrosine in position 639 in T7,or in analogous position in the other polymerases defined above, by aphenylalanine, and/or a mutation corresponding to the substitution ofthe isoleucine in position 810 in T7, or in analogous position in theother polymerases defined above, by a serine, or by an asparagine.

[0064] Therefore, the invention also relates more particularly to theuse of the abovementioned mutated T7 RNA polymerase represented by SEQID NO: 10.

[0065] A subject of the invention is also:

[0066] the mutated RNA polymerases derived from the wild-type T7 RNApolymerase comprising at least one of the abovementioned mutations inposition 117, 119, 134, 147, 230, 266, or 291, and optionally at leastone of the following additional mutations:

[0067] replacement of the tyrosine (Y) in position 639 by aphenylalanine (F),

[0068] replacement of the isoleucine (I) in position 810 by anasparagine (N),

[0069] the mutated RNA polymerases derived from the wild-type T3 RNApolymerase comprising at least one of the abovementioned mutations inposition 148, 267, or 292, and optionally at least one of the followingadditional mutations:

[0070] replacement of the tyrosine (Y) in position 640 by aphenylalanine (F),

[0071] replacement of the isoleucine (I) in position 811 by anasparagine (N),

[0072] the mutated RNA polymerases derived from the wild-type K11 RNApolymerase comprising at least one of the abovementioned mutations inposition 167, 289, or 314, and optionally at least one of the followingadditional mutations:

[0073] replacement of the tyrosine (Y) in position 662 by aphenylalanine (F),

[0074] replacement of the isoleucine (I) in position 833 by anasparagine (N),

[0075] the mutated RNA polymerases derived from the wild-type SP6 RNApolymerase comprising at least one of the abovementioned mutations inposition 117 or 239, and optionally at least one of the followingadditional mutations:

[0076] replacement of the tyrosine (Y) in position 631 by aphenylalanine (F),

[0077] replacement of the isoleucine (I) in position 804 by anasparagine (N).

[0078] The invention also relates to any process for preparing mutatedRNA polymerases of phage origin as defined above, namely of RNApolymerases, the determined nucleotide sequence transcription activityof which, comprising a DNA sequence coding for an RNA of interest andthe transcription of which is placed under the control of a promoterrecognized by the mutated RNA polymerases and by the wild-type RNApolymerases of phage origin from which they originate, is greater thanthe transcription activity of this determined sequence by the wild-typeRNA polymerases, in particular preparation of mutated RNA polymerasesbeing up to approximately 40 times more active than said wild-type RNApolymerases within the scope of the implementation of processes forproducing RNA of interest starting with said determined nucleotidesequence, said process comprising:

[0079] a stage of modification of the peptide chain of the wild-type RNApolymerases of phage origin by substitution, or deletion or addition ofat least one codon of the gene coding for said wild-type RNApolymerases, and transformation of appropriate cells, such as E. coli,with vectors containing the gene thus modified,

[0080] detection of the abovementioned cells producing mutated RNApolymerases for which the production yield of a particular marker withinappropriate cells, such as E. coli, in particular within theabovementioned cells, such as a marker of resistance to an antibiotic,or a chromogenic marker, coded by a nucleotide sequence inserteddownstream of the promoter recognized by the abovementioned RNApolymerases, this promoter and the sequence coding for the marker beingseparated by an ITS, the nature of approximately the first 6 to 12nucleotides of which is known to affect the activity of the wild-typeRNA polymerases, is greater than the production yield of this samemarker obtained by use of the wild-type RNA polymerases under the sameconditions,

[0081] purification of the abovementioned mutated RNA polymerases fromthe cells detected in the preceding stage.

[0082] A more particular subject of the invention is the abovementioneduse of mutated RNA polymerases of phage origin as obtained byimplementation of the preparation process defined above.

[0083] The invention also relates to mutated RNA polymerases of phageorigin as obtained by implementation of a preparation process definedabove.

[0084] A more particular subject of the invention is the mutated RNApolymerases of phage origin as obtained by implementation of apreparation process defined above, originating from the modification ofwild-type phage monomeric polymerases, in particular originating frommonomeric RNA polymerases of bacteriophages such as T7, T3, K11, SP6.

[0085] A subject of the invention is also the abovementioned mutated RNApolymerases of phage origin deriving from the RNA polymerases ofwild-type phage origin, at least one of the amino acids of which,situated between positions 1 and approximately 410, in particularapproximately between positions 90 and 320, more particularly betweenpositions 115 and 300, is modified by substitution or deletion, to theexclusion of the mutated RNA polymerase derived from the wild-type T7RNA polymerase, and comprising the following mutation:

[0086] replacement of the lysine (K) in position 222 by glutamic acid.

[0087] A more particular subject of the invention is the abovementionedmutated RNA polymerases comprising a leucine in position 266,substituted for the proline in position 266 in the wild-type T7 RNApolymerase, or in the homologous positions in the wild-typebacteriophage RNA polymerases, such as positions 267 in T3, 289 in K11,and 239 in SP6.

[0088] A more particular subject of the invention is also any mutatedRNA polymerase as defined above, of which the proline defined above,and/or at least one of the amino acids situated in the vicinity of theabovementioned proline, namely an amino acid situated at a distance lessthan or equal to approximately 10 angströms from the proline inquestion, when said RNA polymerase is considered in itsthree-dimensional structure, is modified by substitution or deletion.

[0089] The invention more particularly relates to the mutated RNApolymerases derived from the wild-type RNA polymerases such as T7, T3,K11 or SP6, and chosen from the following:

[0090] those derived from the wild-type T7 RNA polymerase, andcomprising at least one of the following mutations:

[0091] replacement of the isoleucine (I) in position 117 by a valine(V),

[0092] replacement of the isoleucine (I) in position 119 by a valine(V),

[0093] replacement of the valine (V) in position 134 by an alanine (A),

[0094] replacement of the aspartic acid (D) in position 147 byasparagine (N),

[0095] replacement of the histidine (H) in position 230 by an arginine(R),

[0096] replacement of the proline (P) in position 266 by a leucine (L),

[0097] replacement of the arginine (R) in position 291 by a cysteine(C),

[0098] said mutated RNA polymerases optionally comprising at least oneof the following additional mutations:

[0099] replacement of the tyrosine (Y) in position 639 by aphenylalanine (F),

[0100] replacement of the isoleucine (I) in position 811 by anasparagine (N),

[0101] those derived from the wild-type T3 RNA polymerase, andcomprising at least one of the following mutations:

[0102] replacement of the aspartic acid (D) in position 148 byasparagine (N),

[0103] replacement of the proline (P) in position 267 by a leucine (L),

[0104] replacement of the arginine (R) in position 292 by a cysteine(C),

[0105] said mutated RNA polymerases optionally comprising at least oneof the following additional mutations:

[0106] replacement of the tyrosine (Y) in position 640 by aphenylalanine (F),

[0107] replacement of the isoleucine (I) in position 811 by anasparagine (N),

[0108] those derived from the wild-type K11 RNA polymerase, andcomprising at least one of the following mutations:

[0109] replacement of the aspartic acid (D) in position 167 byasparagine (N),

[0110] replacement of the proline (P) in position 289 by a leucine (L),

[0111] replacement of the arginine (R) in position 314 by a cysteine(C),

[0112] said mutated RNA polymerases optionally comprising at least oneof the following additional mutations:

[0113] replacement of the tyrosine (Y) in position 662 by aphenylalanine (F),

[0114] replacement of the isoleucine (I) in position 833 by anasparagine (N),

[0115] those derived from the wild-type SP6 RNA polymerase, andcomprising at least one of the following mutations:

[0116] replacement of the aspartic acid (D) in position 117 byasparagine (N),

[0117] replacement of the proline (P) in position 239 by a leucine (L),

[0118] said mutated RNA polymerases optionally comprising at least oneof the following additional mutations:

[0119] replacement of the tyrosine (Y) in position 631 by aphenylalanine (F),

[0120] replacement of the isoleucine (I) in position 804 by anasparagine (N).

[0121] A more particular subject of the invention is the followingmutated RNA polymerases:

[0122] the mutated T7 RNA polymerase represented by SEQ ID NO: 2,comprising a leucine in position 266 substituted for the proline,

[0123] the mutated T7 RNA polymerase represented by SEQ ID NO: 4,comprising a valine in position 117 substituted for the isoleucine, andan alanine in position 134 substituted for the valine,

[0124] the mutated T7 RNA polymerase represented by SEQ ID NO: 6,comprising a valine in position 119 substituted for the isoleucine, andan asparagine in position 147 substituted for the aspartic acid,

[0125] the mutated T7 RNA polymerase represented by SEQ ID NO: 8,comprising an arginine in position 230 substituted for the histidine,and a cysteine in position 291 substituted for the arginine,

[0126] the mutated T7 RNA polymerase represented by SEQ ID NO: 10,comprising a leucine in position 266 substituted for the proline, and aphenylalanine in position 639 substituted for the tyrosine,

[0127] the mutated T7 RNA polymerase represented by SEQ ID NO: 12,comprising an asparagine in position 810 substituted for the isoleucine,

[0128] the mutated T7 RNA polymerase represented by SEQ ID NO: 14,comprising a leucine in position 266 substituted for the proline, and anasparagine in position 810 substituted for the isoleucine,

[0129] the mutated T7 RNA polymerase represented by SEQ ID NO: 16,comprising a valine in position 119 substituted for the isoleucine, anasparagine in position 147 substituted for the aspartic acid, and anasparagine in position 810 substituted for the isoleucine.

[0130] The invention also relates to the nucleotide sequences coding fora mutated RNA polymerase of phage origin as defined above.

[0131] Therefore a more particular subject of the invention is thenucleotide sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, codingrespectively for the abovementioned proteins SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, or any nucleotide sequence derived from the abovementionednucleotide sequences by degeneration of the genetic code and retainingthe property of coding for the abovementioned proteins.

[0132] A subject of the invention is also any vector, in particular anyplasmid, containing a nucleotide sequence as defined above.

[0133] A subject of the invention is also any cell transformed by anabovementioned vector, said cell being chosen in particular from thoseof bacteria (E. coli for example), yeasts, or higher eukaryotes.

[0134] The invention also relates to any process for preparing theabovementioned mutated RNA polymerases of phage origin, comprising theculture of transformed cells defined above in an appropriate culturemedium, and the purification of the RNA polymerases produced by saidcells.

[0135] A subject of the invention is also any process for preparing invitro RNA of interest, comprising the bringing together, in anappropriate medium, of at least one mutated RNA polymerase of phageorigin as defined above, with the determined nucleotide sequencescomprising a DNA sequence coding for said RNA of interest and thetranscription of which is placed under the control of a promoterrecognized by the abovementioned wild-type RNA polymerases and mutatedRNA polymerases.

[0136] The invention also relates to any process for preparing in vivoRNA of interest, comprising the culture of cells as defined above, saidcells producing at least one mutated RNA polymerase of phage originaccording to the invention, and the genome of which has been modified inorder to contain determined nucleotide sequences comprising a DNAsequence coding for said RNA of interest and the transcription of whichis placed under the control of a promoter recognized by theabovementioned wild-type RNA polymerases and mutated RNA polymerases.

[0137] A subject of the invention is also any process for preparing invivo proteins of interest comprising the culture of cells as definedabove, said cells producing at least one mutated RNA polymerase of phageorigin according to the invention, and the genome of which has beenmodified in order to contain determined nucleotide sequences comprisinga DNA sequence coding for said proteins of interest and thetranscription of which is placed under the control of a promoterrecognized by the abovementioned wild-type RNA polymerases and mutatedRNA polymerases.

[0138] Advantageously, in the case of the implementation of processesfor preparing proteins of interest as described above, the mutated RNApolymerases produced by the cells, are chosen from SEQ ID NO: 12, 14 and16.

[0139] The invention also relates to any process for preparing in vitroor in vivo RNA or proteins of interest, as described above, andcomprising, moreover, a stage of addition of synthetic polyamines asdescribed by Frugier et al. Amongst the polyamines capable of beingused, the following can be mentioned:

NH₂(CH₂)₃NH(CH₂)₁₀NH(CH₂)₃NH₂

NH₂(CH₂)₃NH(CH₂)₃NH(CH₂)₁₀NH(CH₂)₃NH(CH₂)₃NH₂

[0140] The invention is further illustrated using the following detaileddescription of the production of mutated RNA polymerases of phage originaccording to the invention, and of their use for increasing theproduction of determined RNAs with respect to the production of thesesame RNAs using wild-type RNA polymerases.

[0141] Whilst the RNA polymerase of the bacteriophage T7 is normallyvery processive in the elongation phase, this is not the case during thetranscription of the ITS: in this case, there is a high probability thatthe nascent transcript will be prematurely released (abortivetranscription) (Martin et al., 1988). This is the number of abortivecycles that the enzyme must achieve, on average, before successfullytranscribing the ITS and entering the elongation phase, which determinesthe frequency with which the complete transcripts are synthesized(Ikeda, 1992). This number is minimal when the ITS corresponds to the 5′sequence of the class III genes of the bacteriophage T7 (‘consensual’ITS: 5′ GGGAGA . . . ). However, even under these conditions, theabortive transcripts make up a large proportion (40% to 60%) of thetotal transcripts. This proportion increases very rapidly when the ITSis removed from the consensus, or when the RNA polymerase carriesmutations which reduce its rate of elongation, increasing the timenecessary for the crossing of the ITS (Bonner et al., 1994; Bonner etal., 1992). Optionally, the combination of a non-consensual ITS and aslow polymerase leads to a situation where the enzyme loops indefinitelyin the abortive phase: no large transcript is then synthesized. Thisproperty can be observed not only in vitro, but also in vivo, in cellsof Escherichia coli expressing T7 RNA polymerase (Lopez et al., 1997;Makarova et al., 1995). It is the latter situation that we haveexploited in order to select mutants of T7 RNA polymerase, the activityof which is less sensitive to the nature of the ITS than that of thewild-type enzyme.

[0142] We placed a target gene, the expression of which is easilydetectable—the lacZ gene, the product of which isβ-galactosidase—preceded by a non-consensual ITS, under the control ofthe T7 promoter. This gene is then introduced into a bacterial cellcontaining moreover a plasmid coding for a T7 polymerase mutated in itscatalytic site (i.e. a ‘slow’ polymerase). As indicated above, thesystem, blocked in abortive phase, does not produce large transcripts,and therefore no β-galactosidase. However, after random mutagenesis ofthe plasmid, it is possible to select bacterial clones which synthesizenew mutants of the enzyme which are capable of expressing the targetgene. Apart from the reverse mutants of the initial mutation of thecatalytic site, we thus isolated mutations in the N-terminal part of theenzyme, which is not involved in the catalysis. When these new mutationswere introduced into the wild-type enzyme, i.e. without mutations at thecatalytic site, we observed that they reduced the susceptibility of theenzyme to the exact nature of the ITS. This is the point mutant P266Lwhich is more particularly described here and forms the subject of thepresent invention. In fact, it makes it possible to transcribe, atusable levels, matrices on which the wild-type enzyme is practicallyinoperative.

DETAILED DESCRIPTION

[0143] a) Preparation, purification and storage

[0144] The point mutant SEQ ID NO: 2 obtained corresponds to the changein the sequence of the T7 polymerase of Proline 266 to Leucine (P266L).The gene of this mutated T7 polymerase is included in the pAR1219plasmid under the control of an inducible promoter (pLac UV5). Theprotein (polymerase) is over-expressed in the BL 21 (ompF-) strain ofE.coli. The protocols for extraction, purification and storage of thepurified protein are identical to those of the wild-type polymerase. Inorder to facilitate the purification, the polymerase was labelled withsix N-terminal histidines by transfer of the mutated region in place ofthe equivalent coding sequence of the pBH 161 plasmid. (He et al. 1977).This label in no way modifies the catalytic properties of thepolymerase. It is then sufficient to pass the bacterial supernatant overa Nickel affinity column in order to obtain >90% pure protein. Storagefor a long duration (2 years) at −20° C. does not alter the propertiesof the enzyme.

[0145] b) Catalytic activity.

[0146] Under the standard conditions used for the wild-type polymerase,the mutant is also capable of converting all the rNTPs provided to it toRNA. However the rate at which this incorporation is carried out isapproximately three times lower for the mutant, i.e. a completetranscription will require a longer period of time (e.g. 6 hours insteadof 2 hours).

[0147] As referred to below, the main distinction between the wild-typepolymerase and the mutant is to be found at the level of distribution asa function of the size (and therefore of the mass) of the RNAs obtained.Two types of RNA can be observed on completion of in vitrotranscription: abortive RNAs and large RNAs.

[0148] We present below the in vitro transcription results obtained withthe wild-type polymerase and the mutant on seven very different ITSs.The matrices are plasmid DNAs (approximately 6 kbp) linearized atrestriction sites situated from 31 to 78 nt downstream of the promoter.The reactions are stopped at 10, 20 or 30 minutes depending on the case,and the marking is α³²PGTP, α³²PUTP or γ³²PGTP. The experimentalconditions used are described in J. Mol. Biol. (1997) 269:41-51. Thetranscripts are separated on a high resolution denaturing polyacrylamidegel.

[0149] G10 (5′ GGGAGACCA . . . linearized at 33 nt) carries theconsensual ITS. In fact in this case the first 30 nucleotides correspondto gene 10 sequence of the phage T7, one of those better transcribed byT7 polymerase.

[0150] GGLac (5′ GGGGAAUU. linearized at 69 nt) carries the ITS that isfound in the commercial plasmid pET15b (Novagen) which comprises thebinding site of the Lac repressor in order to optionally repress the T7transcription.

[0151] Lac (5′ GGAAUUG. linearized at 30 nt) is a plasmid equivalent tothe preceding one, in which the operative site is close to 2 nucleotidesof the promoter.

[0152] TyrG (5′ GUCUCGG. linearized at 78 nt), Gly (5′ ACUCUUU.linearized at 71 nt), Tyr (5′ CUCUCGG. linearized at 78 nt) and Val (5′GGUUUCG. linearized at 31 nt) are plasmids corresponding to matricesintended to synthesize in vitro tRNAs or fragments of tRNA and deemeddifficult to transcribe (Frugier et al., 1994)

[0153] FIGS. 1 to 7 show the detailed results indicating the abortiveRNAs observed, their percentage with respect to the total number oftranscripts, as well as the equivalent figures for the long transcriptswhich escaped the abortive phase, in the case of use of wild-type T7polymerase (also designated wt, and represented using black columns) andof the use of the mutated RNA polymerase SEQ ID NO: 2 (also designatedP266L, and represented using grey columns) in the presence of the ITSG10 (FIG. 1), GGLac (FIG. 2), TyrG (FIG. 3), Lac (FIG. 4), Gly (FIG. 5),Tyr (FIG. 6), Val (FIG. 7). The abortive transcripts are distributedbetween the 2 mer and the 13 mer (numbering 2 to 13 along the x-axis).The transcripts originating from polymerases which have escaped theabortive phase are identified as long transcripts (LT along the x-axis).A logarithmic scale is used along the y-axis in order to make itpossible to quantify transcripts which are not very abundant.

[0154] These results show clearly that in all cases the mutant has lessdifficulty than the wild-type polymerase in incorporating the fifth,sixth and seventh nucleotides. If at these positions the polymerase hasand/or must incorporate a pyrimidine (U or C) the abortion rate israised for the wild-type polymerase and the removal of the handicap bythe mutant is all the greater.

[0155] The immediate consequence is summarized in FIG. 8 which includesthe results referred to previously, providing figures comparing theproductivities of the two polymerases for increasingly unfavourableITSs. The values indicated correspond to the percentage of incorporationof the rNTP into large transcripts in the case of use of the wild-typeT7 polymerase (wt, black columns) and the use of the mutated RNApolymerase SEQ ID NO: 2 (P266L, grey columns).

[0156] Apart from G10 and GGLac where the two polymerases give completesatisfaction, in all other cases the wild-type polymerase has a lowyield, even a very low yield, comprised between 22% and 1%, whilst thatof the mutant remains reasonable, being comprised between 30% and 85%.

BIBLIOGRAPHY

[0157] Bonner, G., Lafer, E. M. & Sousa, R. (1994). Characterisation ofa set of T7 RNA polymerase active site mutants. J. Biol. Chem. 269,25120-25128.

[0158] Bonner, G., Patra, D., Lafer, E. M. & Sousa, R. (1992). Mutationsin T7 RNA polymerase which support the proposal for a common polymeraseactive site structure. EMBO J. 11, 3767-3775.

[0159] Davanloo, P., Rosenberg, A. H., Dunn, J. J. & Studier, F. W.(1984). Cloning and expression of the gene for bacteriophage T7 RNApolymerase. Proc. Natl. Acad. Sci. USA 81, 2035-2039.

[0160] Frugier, M., C., F., M. W., H., J-M., L. & R., G. (1994).Synthetic polyamines stimulate in vitro transcription by T7 RNApolymerase. Nucl. Acid Res. 22, 2784-2790.

[0161] He, B., Rong, M., Lyakhov, D., Gartenstein, H., Diaz, G.,Castagna, R., McAllister, W. T. & Durbin, R. K. (1997). Rapidmutagenesis and purification of phage RNA polymerase. Protein ExpressPurif. 9.142-151.

[0162] Ikeda, R. A. (1992). The efficiency of promoter clearancedistinguishes T7 class II and class III promoters. J. Biol. Chem. 267,11322-11328.

[0163] Lapham J, Yu Y T, Shu M D, Steitz J A, Crothers D M.(1997) Theposition of site-directed cleavage of RNA using RNase H and 2′-O-methyloligonucleotides is dependent on the enzyme source. RNA 3.950-951

[0164] LiZ, Pandit S, Deutscher M P. (1999). Maturation of 23S ribosomalRNA requires the exorbonuclease RNase T. RNA 5.139-146

[0165] Lopez, P. J., Guillerez, J., Sousa, R. & Dreyfus, M. (1997). Thelow processivity of T7 RNA polymerase over the initially transcribedsequence can limit productive initiation in vivo. J. Mol. Biol. 269,41-51.

[0166] Makarova, O. V., Makarov, E. M., Sousa, R. & Dreyfus, M. (1995).Transcribing Escherichia coli genes with mutant T7 RNA polymerases:stability of lacZ mRNA inversely correlates with polymerase speed. Proc.Natl. Acad. Sci. USA 92, 12250-12254.

[0167] Martin, C. T., Muller, D. K. & Coleman, J. E. (1988).Processivity in early stages of transcription by T7 RNA polymerase.Biochemistry 27, 3966-3974.

[0168] Milligan, J. F., Groebe, D. R., Witherell, G. W. & Uhlenbeck, O.C. (1987). Oligonucleotide synthesis using T7 RNA polymerase andsynthetic DNA templates. Nucleic Acids Res. 15(21), 8783-8798.

1 16 1 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 1 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac atc accatt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Ile Thr IleLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gttcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Val GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag gac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asp Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacac cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu HisArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ctg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Leu Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt cgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Arg Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct tac ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg att cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 2 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 2 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile ThrIle Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrVal Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Leu Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla 3 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 3 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac gtc accatt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Val Thr IleLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gctcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Ala GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag gac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asp Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacac cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu HisArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ccg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt cgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Arg Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct tac ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg att cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 4 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 4 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Val ThrIle Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrAla Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla 5 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 5 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac atc accgtt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Ile Thr ValLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gttcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Val GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag aac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asn Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacac cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu HisArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ccg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt cgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Arg Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct tac ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg att cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 6 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 6 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile ThrVal Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrVal Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asn GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla 7 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 7 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac atc accatt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Ile Thr IleLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gttcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Val GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag gac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asp Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacgc cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu ArgArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ccg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt tgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Cys Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct tac ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg att cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 8 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 8 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile ThrIle Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrVal Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu Arg Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Cys Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla 9 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 9 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac atc accatt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Ile Thr IleLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gttcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Val GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag gac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asp Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacac cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu HisArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ctg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Leu Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt cgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Arg Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct ttc ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Phe Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg att cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 10 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 10 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile ThrIle Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrVal Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Leu Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Phe Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla 11 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 11 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac atc accatt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Ile Thr IleLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gttcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Val GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag gac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asp Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacac cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu HisArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ccg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt cgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Arg Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct tac ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg aat cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Asn His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 12 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 12 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile ThrIle Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrVal Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Asn His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla 13 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 13 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac atc accatt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Ile Thr IleLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gttcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Val GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag gac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asp Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacac cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu HisArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ctg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Leu Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt cgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Arg Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct tac ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg aat cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Asn His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 14 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 14 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile ThrIle Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrVal Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Leu Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Asn His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla 15 2652 DNA Artificial sequence Description of the artificialsequence mutated sequences of bacteriophage T7 RNA polymerase 15 atg aacacg att aac atc gct aag aac gac ttc tct gac atc gaa ctg 48 Met Asn ThrIle Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 gct gctatc ccg ttc aac act ctg gct gac cat tac ggt gag cgt tta 96 Ala Ala IlePro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 gct cgc gaacag ttg gcc ctt gag cat gag tct tac gag atg ggt gaa 144 Ala Arg Glu GlnLeu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 gca cgc ttc cgcaag atg ttt gag cgt caa ctt aaa gct ggt gag gtt 192 Ala Arg Phe Arg LysMet Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 gcg gat aac gct gccgcc aag cct ctc atc act acc cta ctc cct aag 240 Ala Asp Asn Ala Ala AlaLys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 atg att gca cgc atcaac gac tgg ttt gag gaa gtg aaa gct aag cgc 288 Met Ile Ala Arg Ile AsnAsp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 ggc aag cgc ccg aca gccttc cag ttc ctg caa gaa atc aag ccg gaa 336 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 gcc gta gcg tac atc accgtt aag acc act ctg gct tgc cta acc agt 384 Ala Val Ala Tyr Ile Thr ValLys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 gct gac aat aca acc gttcag gct gta gca agc gca atc ggt cgg gcc 432 Ala Asp Asn Thr Thr Val GlnAla Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 att gag aac gag gct cgcttc ggt cgt atc cgt gac ctt gaa gct aag 480 Ile Glu Asn Glu Ala Arg PheGly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 cac ttc aag aaa aacgtt gag gaa caa ctc aac aag cgc gta ggg cac 528 His Phe Lys Lys Asn ValGlu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 gtc tac aag aaa gcattt atg caa gtt gtc gag gct gac atg ctc tct 576 Val Tyr Lys Lys Ala PheMet Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 aag ggt cta ctc ggtggc gag gcg tgg tct tcg tgg cat aag gaa gac 624 Lys Gly Leu Leu Gly GlyGlu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 tct att cat gta ggagta cgc tgc atc gag atg ctc att gag tca acc 672 Ser Ile His Val Gly ValArg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 gga atg gtt agc ttacac cgc caa aat gct ggc gta gta ggt caa gac 720 Gly Met Val Ser Leu HisArg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 tct gag act atcgaa ctc gca cct gaa tac gct gag gct atc gca acc 768 Ser Glu Thr Ile GluLeu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 cgt gca ggt gcgctg gct ggc atc tct ccg atg ttc caa cct tgc gta 816 Arg Ala Gly Ala LeuAla Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 gtt cct cct aagccg tgg act ggc att act ggt ggt ggc tat tgg gct 864 Val Pro Pro Lys ProTrp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 aac ggt cgt cgtcct ctg gcg ctg gtg cgt act cac agt aag aaa gca 912 Asn Gly Arg Arg ProLeu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 ctg atg cgc tacgaa gac gtt tac atg cct gag gtg tac aaa gcg att 960 Leu Met Arg Tyr GluAsp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 aac att gcgcaa aac acc gca tgg aaa atc aac aag aaa gtc cta gcg 1008 Asn Ile Ala GlnAsn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 gtc gcc aacgta atc acc aag tgg aag cat tgt ccg gtc gag gac atc 1056 Val Ala Asn ValIle Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 cct gcg attgag cgt gaa gaa ctc ccg atg aaa ccg gaa gac atc gac 1104 Pro Ala Ile GluArg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 atg aat cctgag gct ctc acc gcg tgg aaa cgt gct gcc gct gct gtg 1152 Met Asn Pro GluAla Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 tac cgc aaggac aag gct cgc aag tct cgc cgt atc agc ctt gag ttc 1200 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 atg cttgag caa gcc aat aag ttt gct aac cat aag gcc atc tgg ttc 1248 Met Leu GluGln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 cct tacaac atg gac tgg cgc ggt cgt gtt tac gct gtg tca atg ttc 1296 Pro Tyr AsnMet Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 aac ccgcaa ggt aac gat atg acc aaa gga ctg ctt acg ctg gcg aaa 1344 Asn Pro GlnGly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 ggt aaacca atc ggt aag gaa ggt tac tac tgg ctg aaa atc cac ggt 1392 Gly Lys ProIle Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 gca aactgt gcg ggt gtc gat aag gtt ccg ttc cct gag cgc atc aag 1440 Ala Asn CysAla Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 ttcatt gag gaa aac cac gag aac atc atg gct tgc gct aag tct cca 1488 Phe IleGlu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 ctggag aac act tgg tgg gct gag caa gat tct ccg ttc tgc ttc ctt 1536 Leu GluAsn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 gcgttc tgc ttt gag tac gct ggg gta cag cac cac ggc ctg agc tat 1584 Ala PheCys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 aactgc tcc ctt ccg ctg gcg ttt gac ggg tct tgc tct ggc atc cag 1632 Asn CysSer Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 cacttc tcc gcg atg ctc cga gat gag gta ggt ggt cgc gcg gtt aac 1680 His PheSer Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560ttg ctt cct agt gaa acc gtt cag gac atc tac ggg att gtt gct aag 1728 LeuLeu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575aaa gtc aac gag att cta caa gca gac gca atc aat ggg acc gat aac 1776 LysVal Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590gaa gta gtt acc gtg acc gat gag aac act ggt gaa atc tct gag aaa 1824 GluVal Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605gtc aag ctg ggc act aag gca ctg gct ggt caa tgg ctg gct tac ggt 1872 ValLys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620gtt act cgc agt gtg act aag cgt tca gtc atg acg ctg gct tac ggg 1920 ValThr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635640 tcc aaa gag ttc ggc ttc cgt caa caa gtg ctg gaa gat acc att cag 1968Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650655 cca gct att gat tcc ggc aag ggt ctg atg ttc act cag ccg aat cag 2016Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665670 gct gct gga tac atg gct aag ctg att tgg gaa tct gtg agc gtg acg 2064Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 gtg gta gct gcg gtt gaa gca atg aac tgg ctt aag tct gct gct aag 2112Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695700 ctg ctg gct gct gag gtc aaa gat aag aag act gga gag att ctt cgc 2160Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710715 720 aag cgt tgc gct gtg cat tgg gta act cct gat ggt ttc cct gtg tgg2208 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725730 735 cag gaa tac aag aag cct att cag acg cgc ttg aac ctg atg ttc ctc2256 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740745 750 ggt cag ttc cgc tta cag cct acc att aac acc aac aaa gat agc gag2304 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755760 765 att gat gca cac aaa cag gag tct ggt atc gct cct aac ttt gta cac2352 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770775 780 agc caa gac ggt agc cac ctt cgt aag act gta gtg tgg gca cac gag2400 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785790 795 800 aag tac gga atc gaa tct ttt gca ctg aat cac gac tcc ttc ggtacc 2448 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Asn His Asp Ser Phe Gly Thr805 810 815 att ccg gct gac gct gcg aac ctg ttc aaa gca gtg cgc gaa actatg 2496 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met820 825 830 gtt gac aca tat gag tct tgt gat gta ctg gct gat ttc tac gaccag 2544 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln835 840 845 ttc gct gac cag ttg cac gag tct caa ttg gac aaa atg cca gcactt 2592 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu850 855 860 ccg gct aaa ggt aac ttg aac ctc cgt gac atc tta gag tcg gacttc 2640 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880 gcg ttc gcg taa 2652 Ala Phe Ala 16 883 PRT Artificialsequence Description of the artificial sequence mutated sequences ofbacteriophage T7 RNA polymerase 16 Met Asn Thr Ile Asn Ile Ala Lys AsnAsp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr LeuAla Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu GluHis Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe GluArg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys ProLeu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn AspTrp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala PheGln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile ThrVal Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr ThrVal Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asn GluAla Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His PheLys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 ValTyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala IleAla Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe GlnPro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly GlyGly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg ThrHis Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met ProGlu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala TrpLys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr LysTrp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg GluGlu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu AlaLeu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys AspLys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met LeuGlu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 ProTyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala LysSer Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro PheCys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His HisGly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly SerCys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu ValGly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val GlnAsp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu GlnAla Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val ThrAsp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly ThrLys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg SerVal Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser LysGlu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 ProAla Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe ProVal Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn LeuMet Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr AsnLys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile AlaPro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys ThrVal Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe AlaLeu Asn His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala AsnLeu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu SerCys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln LeuHis Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys GlyAsn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala PheAla

1. Use of mutated RNA polymerases of phage origin, the peptide chain ofwhich is modified, with respect to the wild-type RNA polymerases fromwhich they originate, by substitution, or deletion, or addition of atleast one amino acid, this modification having the effect of reducingthe sensitivity of said RNA polymerases to the ITS contained in the DNAsequence coding for an RNA of interest, for the implementation of aprocess for producing said RNA of interest, or proteins coded by thisRNA of interest, starting from determined nucleotide sequencescomprising a DNA sequence coding for said RNA of interest and thetranscription of which is placed under the control of a promoterrecognized by the abovementioned wild-type RNA polymerases and mutatedRNA polymerases, said process having a production yield of said RNAgreater than the yield obtained in the case of use of the wild-type RNApolymerases in the presence of the same non-consensual ITS as thatcontained in the DNA sequence coding for said RNA of interest.
 2. Useaccording to claim 1, for the implementation of a process for producingRNA of interest, the yield of which is up to approximately 10 timesgreater than the yield obtained in the case of use of the wild-type RNApolymerases.
 3. Use according to claim 1 or 2, of mutated RNApolymerases originating from wild-type phage monomeric polymerases, inparticular originating from monomeric RNA polymerases of bacteriophagessuch as T7, T3, K11, SP6.
 4. Use of mutated RNA polymerases according toone of claims 1 to 3, derived from the wild-type RNA polymerases atleast one of the amino acids of which, situated between positions 1 andapproximately 410, or approximately between positions 90 and 320, orbetween positions 115 and 300, is modified by substitution or deletion.5. Use according to one of claims 1 to 4, of mutated RNA polymeraseschosen from: those comprising a leucine in position 266, substituted forthe proline situated in position 266 in the wild-type T7 RNA polymerase,or of the proline situated in homologous position in the wild-type RNApolymerases of bacteriophages, such as the proline situated in positions267 in T3, 289 in K11, and 239 in SP6, and/or those at least one of theamino acids of which, situated in the vicinity of the abovementionedproline, namely an amino acid situated at a distance less than or equalto approximately 10 angströms from the proline in question, is modifiedby substitution or deletion.
 6. Use according to one of claims 1 to 5 ofthe following mutated RNA polymerases: those derived from the wild-typeT7 RNA polymerase, and comprising at least one of the followingmutations: replacement of the isoleucine (I) in position 117 by a valine(V), replacement of the isoleucine (I) in position 119 by a valine (V),replacement of the valine (V) in position 134 by an alanine (A),replacement of the aspartic acid (D) in position 147 by asparagine (N),replacement of the histidine (H) in position 230 by an arginine (R),replacement of the proline (P) in position 266 by a leucine (L),replacement of the arginine (R) in position 291 by a cysteine (C), saidmutated RNA polymerases optionally comprising at least one of thefollowing additional mutations: replacement of the tyrosine (Y) inposition 639 by a phenylalanine (F), replacement of the isoleucine (I)in position 810 by an asparagine (N), those derived from the wild-typeT3 RNA polymerase, and comprising at least one of the followingmutations: replacement of the aspartic acid (D) in position 148 byasparagine (N), replacement of the proline (P) in position 267 by aleucine (L), replacement of the arginine (R) in position 292 by acysteine (C), said mutated RNA polymerases optionally comprising atleast one of the following additional mutations: replacement of thetyrosine (Y) in position 640 by a phenylalanine (F), replacement of theisoleucine (I) in position 811 by an asparagine (N), those derived fromthe wild-type K11 RNA polymerase, and comprising at least one of thefollowing mutations: replacement of the aspartic acid (D) in position167 by asparagine (N), replacement of the proline (P) in position 289 bya leucine (L), replacement of the arginine (R) in position 314 by acysteine (C), said mutated RNA polymerases optionally comprising atleast one of the following additional mutations: replacement of thetyrosine (Y) in position 662 by a phenylalanine (F), replacement of theisoleucine (I) in position 833 by an asparagine (N), those derived fromthe wild-type SP6 RNA polymerase, and comprising at least one of thefollowing mutations: replacement of the aspartic acid (D) in position117 by asparagine (N), replacement of the proline (P) in position 239 bya leucine (L), said mutated RNA polymerases optionally comprising atleast one of the following additional mutations: replacement of thetyrosine (Y) in position 631 by a phenylalanine (F), replacement of theisoleucine (I) in position 804 by an asparagine (N).
 7. Use according toone of claims 1 to 6 of the following mutated RNA polymerases: themutated T7 RNA polymerase represented by SEQ ID NO: 2, comprising aleucine in position 266 substituted for the proline, the mutated T7 RNApolymerase represented by SEQ ID NO: 4, comprising a valine in position117 substituted for the isoleucine, and an alanine in position 134substituted for the valine, the mutated T7 RNA polymerase represented bySEQ ID NO: 6, comprising a valine in position 119 substituted for theisoleucine, and an asparagine in position 147 substituted for theaspartic acid, the mutated T7 RNA polymerase represented by SEQ ID NO:8, comprising an arginine in position 230 substituted for the histidine,and a cysteine in position 291 substituted for the arginine, the mutatedT7 RNA polymerase represented by SEQ ID NO: 10, comprising a leucine inposition 266 substituted for the proline, and a phenylalanine inposition 639 substituted for the tyrosine, the mutated T7 RNA polymeraserepresented by SEQ ID NO: 12, comprising an asparagine in position 810substituted for the isoleucine, the mutated T7 RNA polymeraserepresented by SEQ ID NO: 14, comprising a leucine in position 266substituted for the proline, and an asparagine in position 810substituted for the isoleucine, the mutated T7 RNA polymeraserepresented by SEQ ID NO: 16, comprising a valine in position 119substituted for the isoleucine, an asparagine in position 147substituted for the aspartic acid, and an asparagine in position 810substituted for the isoleucine.
 8. Process for preparing mutated RNApolymerases of phage origin, of which the determined nucleotide sequencetranscription activity, comprising a DNA sequence coding for an RNA ofinterest and the transcription of which is placed under the control of apromoter recognized by the mutated RNA polymerases and by the wild-typeRNA polymerases from which they originate, is greater than thetranscription activity of this determined sequence by the wild-type RNApolymerases, in particular for preparing mutated RNA polymerases beingup to approximately 40 times more active than said wild-type RNApolymerases within the scope of the implementation of processes forproducing RNA of interest from said determined nucleotide sequence, saidprocess comprising: a stage of modification of the peptide chain of thewild-type RNA polymerases of phage origin by substitution, or deletionor addition of at least one codon of the gene coding for said wild-typeRNA polymerases, and transformation of appropriate cells, such as E.coli, with vectors containing the gene thus modified, detection of theabovementioned cells producing mutated RNA polymerases for which theproduction yield of a particular marker within appropriate cells, suchas E. coli, in particular within the abovementioned cells, such as amarker of resistance to an antibiotic, or a chromogenic marker, coded bya nucleotide sequence inserted downstream of the promoter recognized bythe abovementioned RNA polymerases, this promoter and the sequencecoding for the marker being separated by an ITS the nature of the firstapproximately 6 to 12 nucleotides of which is known to affect theactivity of the wild-type RNA polymerases, is greater than theproduction yield of this same marker obtained by use of the wild-typeRNA polymerases under the same conditions, purification of theabovementioned mutated RNA polymerases from the cells detected in thepreceding stage.
 9. Use according to one of claims 1 to 7, of mutatedRNA polymerases of phage origin as obtained by implementation of aprocess according to claim
 8. 10. Mutated RNA polymerases of phageorigin as obtained by implementation of a process according to claim 8.11. Mutated RNA polymerases of phage origin according to claim 10,originating from the modification of wild-type phage monomericpolymerases, in particular originating from monomeric RNA polymerases ofbacteriophages such as T7, T3, K11, SP6.
 12. Mutated RNA polymerases ofphage origin deriving from the wild-type RNA polymerases of phage originat least one of the amino acids of which, situated between positions 1and approximately 410, or approximately between positions 90 and 320, orbetween positions 115 and 300, is modified by substitution or deletion,to the exclusion of the mutated RNA polymerase derived from thewild-type T7 RNA polymerase, and comprising the following mutation:replacement of the lysine (K) in position 222 by glutamic acid. 13.Mutated RNA polymerases of phage origin according to claim 12, chosenfrom: those comprising a leucine in position 266, substituted for theproline situated in position 266 in the wild-type T7 RNA polymerase, orfor the proline situated in homologous position in the wild-type RNApolymerases of bacteriophages, such as the proline situated in positions267 in T3, 289 in K11, and 239 in SP6, and/or those at least one of theamino acids of which, situated in the vicinity of the abovementionedproline, namely an amino acid situated at a distance less than or equalto approximately 10 angströms from the proline in question, is modifiedby substitution or deletion.
 14. Mutated RNA polymerases chosen from thefollowing: those derived from the wild-type T7 RNA polymerase, andcomprising at least one of the following mutations: replacement of theisoleucine (I) in position 117 by a valine (V), replacement of theisoleucine (I) in position 119 by a valine (V), replacement of thevaline (V) in position 134 by an alanine (A), replacement of theaspartic acid (D) in position 147 by asparagine (N), replacement of thehistidine (H) in position 230 by an arginine (R), replacement of theproline (P) in position 266 by a leucine (L), replacement of thearginine (R) in position 291 by a cysteine (C), said mutated RNApolymerases optionally comprising at least one of the followingadditional mutations: replacement of the tyrosine (Y) in position 639 bya phenylalanine (F), replacement of the isoleucine (I) in position 810by an asparagine (N), those derived from the wild-type T3 RNApolymerase, and comprising at least one of the following mutations:replacement of the aspartic acid (D) in position 148 by asparagine (N),replacement of the proline (P) in position 267 by a leucine (L),replacement of the arginine (R) in position 292 by a cysteine (C), saidmutated RNA polymerases optionally comprising at least one of thefollowing additional mutations: replacement of the tyrosine (Y) inposition 640 by a phenylalanine (F), replacement of the isoleucine (I)in position 811 by an asparagine (N), those derived from the wild-typeK11 RNA polymerase, and comprising at least one of the followingmutations: replacement of the aspartic acid (D) in position 167 byasparagine (N), replacement of the proline (P) in position 289 by aleucine (L), replacement of the arginine (R) in position 314 by acysteine (C), said mutated RNA polymerases optionally comprising atleast one of the following additional mutations: replacement of thetyrosine (Y) in position 662 by a phenylalanine (F), replacement of theisoleucine (I) in position 833 by an asparagine (N), those derived fromthe wild-type SP6 RNA polymerase, and comprising at least one of thefollowing mutations: replacement of the aspartic acid (D) in position117 by asparagine (N), replacement of the proline (P) in position 239 bya leucine (L), said mutated RNA polymerases optionally comprising atleast one of the following additional mutations: replacement of thetyrosine (Y) in position 631 by a phenylalanine (F), replacement of theisoleucine (I) in position 804 by an asparagine (N).
 15. Mutated RNApolymerases chosen from the following: the mutated T7 RNA polymeraserepresented by SEQ ID NO: 2, comprising a leucine in position 266substituted for the proline, the mutated T7 RNA polymerase representedby SEQ ID NO: 4, comprising a valine in position 117 substituted for theisoleucine, and an alanine in position 134 substituted for the valine,the mutated T7 RNA polymerase represented by SEQ ID NO: 6, comprising avaline in position 119 substituted for the isoleucine, and an asparaginein position 147 substituted for the aspartic acid, the mutated T7 RNApolymerase represented by SEQ ID NO: 8, comprising an arginine inposition 230 substituted for the histidine, and a cysteine in position291 substituted for the arginine, the mutated T7 RNA polymeraserepresented by SEQ ID NO: 10, comprising a leucine in position 266substituted for the proline, and a phenylalanine in position 639substituted for the tyrosine, the mutated T7 RNA polymerase representedby SEQ ID NO: 12, comprising an asparagine in position 810 substitutedfor the isoleucine, the mutated T7 RNA polymerase represented by SEQ IDNO: 14, comprising a leucine in position 266 substituted for theproline, and an asparagine in position 810 substituted for theisoleucine, the mutated T7 RNA polymerase represented by SEQ ID NO: 16,comprising a valine in position 119 substituted for the isoleucine, anasparagine in position 147 substituted for the aspartic acid, and anasparagine in position 810 substituted for the isoleucine. 16.Nucleotide sequence coding for an RNA polymerase according to one ofclaims 10 to
 15. 17. Nucleotide sequences according to claim 16 chosenfrom the sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, codingrespectively for the proteins SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, orany nucleotide sequence derived from the abovementioned nucleotidesequences by degeneration of the genetic code and retaining the propertyof coding for the abovementioned proteins.
 18. Vector containing anucleotide sequence according to claim
 16. 19. Cell transformed by avector according to claim
 18. 20. Process for preparing in vitro RNA ofinterest, comprising the bringing together, in an appropriate medium, ofat least one mutated RNA polymerase according to one of claims 10 to 15,with the determined nucleotide sequences comprising a DNA sequencecoding for said RNA of interest and the transcription of which is placedunder the control of a promoter recognized by the abovementionedwild-type RNA polymerases and mutated RNA polymerases.
 21. Process forpreparing in vivo RNA of interest, comprising the culture of cellsaccording to claim 19, said cells producing at least one mutated RNApolymerase according to one of claims 10 to 15, and the genome of whichhas been modified in order to contain determined nucleotide sequencescomprising a DNA sequence coding for said RNA of interest and thetranscription of which is placed under the control of a promoterrecognized by the abovementioned wild-type RNA polymerases and mutatedRNA polymerases.
 22. Process for preparing in vivo proteins of interestcomprising the culture of cells according to claim 19, said cellsproducing at least one mutated RNA polymerase according to one of claims10 to 15, and the genome of which has been modified in order to containdetermined nucleotide sequences comprising a DNA sequence coding forsaid proteins of interest and the transcription of which is placed underthe control of a promoter recognized by the abovementioned wild-type RNApolymerases and mutated RNA polymerases.
 23. Process for preparing invitro or in vivo RNA or proteins of interest, according to one of claims20 to 22, also comprising a stage of addition of synthetic polyamines.